Next Generation Sequencing Facilitates Quantitative Analysis of WT and WT+CXCL14 mouse endothelial cells' Transcriptomes in mammary gland

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to analysis the differiational genes and pathways in WT and WT+CXCL14 mammary endothelial cells by using NGS-derived transcriptome profiling (RNA-seq). Methods: Recombinant mouse CXCL14 protein in 50 μL of PBS (50μg/kg) was injected into the tail vein every 3 days for 2 weeks. The mice in the control group were treated with an identical volume of PBS. The mice were sacrificed by excessive anaesthesia 14 days after cytokine administration. Mammary glands of 6-week-old female virgin mice were harvested and single-cell suspension was obtained by Collagenase III-mediated digestion of minced tissue, followed by trypsin-EDTA and DNaseI sequential incubation before filtering through 70 μm cell strainers. The following antibodies in 1:200 dilutions were used: rat anti-mouse CD16/CD32, FITC-conjugated hematopoietic lineage cocktail, APC-conjugated CD31. All analysis and sorting were performed using a FACS Aria III sorter. The purity of sorted population was routinely checked and ensured to be > 95%. WT and WT+CXCL14 endothelial cells' mRNA profiles were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with following methods: Alignment by using HISAT2 v2.1, IGV was used to to view the mapping result by the Heatmap, histogram, scatter plot or other stytle, FPKM was then calculated to estimate the expression level of genes in each sample, DEGseq v1.18.0 was used for differential gene expression analysis between two samples with non biological replicates and Function Enrichment Analysis including GO enrichment analysis and KEGG . Conclusions: Our study represents the first detailed analysis of WT and WT+CXCL14 endothelial cells' transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

ORGANISM(S): Mus musculus

PROVIDER: GSE207137 | GEO | 2022/10/08

REPOSITORIES: GEO